JP7390305B2 - Microfluidic chip device for optical power measurement and cell imaging using microfluidic chip configuration and dynamics - Google Patents

Description

The present invention relates generally to devices and methods for particle analysis and imaging of particles or cells in fluids, and more particularly to particle imaging of fluids using pressure, fluid dynamics, electrokinetics, and optical forces. Relating to a device and method for.

The present invention is a microfluidic system in which injection is performed in an upwardly perpendicular direction and fluidic vials are placed below the chip in order to minimize sedimentation of particles in front of and in the analysis part of the channels of the chip. Regarding chips.

In order to implement the present invention, changes had to be made to existing microfluidic chip designs. For example, in order to keep the vial vertically in-line with the chip, a different interface with the chip had to be established compared to the prior art. Specifically, instead of an input tube interfacing with the chip through a port attached to the largest side of the chip (as is typically done in microfluidic lab-on-a-chip systems), we first Fluid is then pumped across the chip and the chip is then raised. The invention, in one aspect, uses a manifold to have the input and fluid rise through the bottom of the chip, thereby avoiding horizontal reorientation of the fluid/hydrodynamics.

According to the invention, the contents of the vial are placed below the chip and pumped directly upward and vertically into the first channel of the chip. A long channel extends from the bottom of the chip to near the top of the chip. Then, the channel takes a short horizontal turn, but the new channel is very short, almost negating the effects of cell sedimentation due to gravity and zero flow velocity at the walls. The fluid is then pumped to the analysis section, unlike the prior art. Therefore, the horizontal analysis part is the highest channel/fluid point in the chip and is therefore closer to the top of the chip, resulting in less chip material (e.g. glass) between the microscope/camera and the sample than in the prior art. less and therefore sharper imaging is obtained. The laser also suspends the cells within this channel during analysis, preventing them from settling.

According to the prior art, microfluidic chip vials containing cells or particles to be separated and/or analyzed are placed on the side and pumped horizontally into channels within the microfluidic chip. First, the contents of the vial (e.g., particles or cells) were pumped vertically upwards, then made a U-turn to move downwards, and then pumped horizontally into the chip (e.g. , U.S. Pat. No. 9,594,071).

The connection to the chip is horizontal and this, combined with the dead volume in the connection (empty space in the fluidic connection which is somewhat unavoidable), results in significant additional settling due to gravity. Such configurations also require relatively large diameter channels required by the connections, which, in addition to dead volumes, create regions of relatively low velocity, further increasing the problem of particle settling. The current tip according to the invention eliminates the need for a large horizontal input channel and a rather abrupt change from the large horizontal input channel to a relatively thin upward flow in the first vertical tip channel. Such a configuration eliminates unnecessary directional changes that cause horizontal settling and subsidence. Allowing the cells to enter the bottom edge of the chip places it perpendicular to gravity so that the cells or particles cannot settle to the bottom of the horizontal channel, but rather they are always guided upwards by the flow. Orientation also solves the problem of settling into dead volumes. This is not intuitive and requires a lot of experimentation to realize the problem before designing the current concrete solution. Currently available microfluidic devices, in contrast to the present invention, incorporate custom or commercially available connections on polished surfaces and larger areas of glass, which generally allow for any Particles (eg, cells) are forced to immediately rotate and move horizontally once they enter the chip.

Also, in the prior art, cells or particles have several horizontal runs on the microfluidic chip before reaching the analysis channel, which results in sedimentation. Once the vial contents enter the chip and the channels within the chip, the contents are pumped horizontally compared to the vertical in-chip channels. The channel then flows upwards and takes a long horizontal turn. At this point, gravity tends to cause the cells to settle to the bottom of the channel. You will also experience lower velocities at the walls due to laminar flow conditions. Essentially, due to the parabolic velocity profile, the flow is highest in the middle of the channel and decreases to zero at or near the channel wall. After the first horizontal in-chip channel, the fluid takes a downward turn before the analysis channel where particles are imaged or separated. Because of this configuration, there is a relatively large distance between the microscope/camera and the analysis channel. In this typical prior art configuration, particles are forced downward and eventually exit the bottom of the chip.

Additionally, due to limitations of the prior art, multiple horizontal runs are required to settle cells at multiple locations within the channel. This in turn causes a reduction in image quality as it is now necessary to image through additional material at the edge of the chip. Prior art channels within the chip are pumped vertically upwards, then horizontally, then in a zigzag nature for appropriate cell or particle suspensions, then under the chip, and out of the chip. had to be done. Zigzag channels are eliminated by the present invention.

Prior art also exists for rendering 3D images of cells or particles in fluids. For example, M. Habaza, M. Kirschbaum, C. Guernth-Marschner, G. Dardikman, I. Barnea, R. Korenstein, C. Duschl, N. T. Shaked, Adv. Sci., 2017, 4, 1600205, teaches capturing a cell, rotating it at high speed, and using interferometry to measure the refractive index distribution within the cell. Interferometry has also been used to analyze cells in microfluidic channels (see, eg, YSung et al., Phys. Rev. Appl. February 27, 2014, 1:014002). However, the present invention claims to take multiple images of cells or particles as they move through the fluid flow and pass through the focal plane of an imaging device, thereby providing a 3D image. Disable the need to capture cells to render. Other techniques have been taught, such as using mechanical translation stages to move cells or particles (e.g., NLue et al., Opt. Express, September 29, 2008, S16(20): 16240-6). ), none of them use bright-field imaging as described herein and do not utilize fluid flow to provide cell positioning relative to the image focal plane.

All prior art documents cited herein are incorporated by reference in their entirety.

The present invention relates to a microfluidic chip in which injection is performed and a sample vial is placed below the chip to minimize particle sedimentation. The contents of the vial are thus located below the chip and are pumped directly upward and vertically into the channels of the chip. A long channel extends from the bottom of the chip to near the top of the chip. The channel then takes a short horizontal turn, but the new channel is short enough to be insignificant with respect to the effect of cell settling due to zero flow velocity at the channel walls. Then, contrary to the conventional technique, the sample is pumped up to the analysis section. Therefore, the horizontal analysis part is the highest channel/fluid point in the chip and is therefore closer to the top of the chip, resulting in less glass between the microscope/camera than in the prior art and therefore clearer imaging. It will be done. The distance of the analysis channel from the top of the chip can be 100 microns to 2 mm, but can be as large as 100 mm, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns. In one embodiment, after the analysis portion of the chip, the sample, eg, fluid, cells, and/or particles, is pumped down to the bottom of the chip and forced outward.

The present invention is further directed to a microfluidic chip in which horizontal flow is minimized, particularly at the point where fluid enters the chip channels. The prior art chip contains a horizontal channel (non-analysis part) of approximately 13 mm, and its injection port is much larger in diameter, approximately 2 mm, exacerbating sedimentation due to low velocity. The chips described herein have horizontal channels (non-analytical) in preferred embodiments with lengths of about 0.2 to 3.0 mm, but horizontal channels of length from 0.01 to 100.0 mm, such as from 0.01 mm to It can range from 0.02 mm, 0.02 mm to 0.03 mm, 0.03 mm to 0.04 mm, etc. This is a result of the channel system of the present invention, which is an order of magnitude different from the prior art, improving cell/particle flow and eliminating sedimentation.

Another aspect of the invention relates to a microfluidic chip in which imaging is performed and analysis is based at or near a corner of the chip, whereby less glass and distance is present between the camera and the analysis channel; Imaging from multiple perspectives is improved. This also allows the use of higher numerical objectives to improve detailed imaging by increasing the magnification. Such design improvements reduce image distortion caused by glass (or other materials that make up the chip, such as plastic or any transparent or translucent material) and distance (e.g., due to defects in the glass). The distance between the imaging device and the analysis channel may be 100 microns to 2 mm, but may be as large as 100 mm, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns. Good too.

Furthermore, the present invention relates to a microfluidic sorting chip that is separated downstream from the analysis channel and that uses both pressure and/or laser (or other optical power) simultaneously or sequentially to actuate the sorting function. enable. In one aspect, flow continues from the analysis channel for the classification function. For example, particles are directed into a vertical channel and then into a horizontal sorting channel. In one embodiment, optical force and/or pressure is applied in the direction of flow using a sorting channel to force particles through the channel. Particles that are not directly acted upon by optical forces may be affected by, for example, gravity, electromotive forces, magnetic forces, laminar flow lines, flow lines, reduced flow rates, orthogonal optical forces, or vacuum applied to draw the particles into alternative channels. detours to an alternate channel. In another aspect related to the post-sort analysis channel, the present invention directs the optical force from the back side of the chip (the laser or optical force is directed in the same flow direction), and in some embodiments this primary laser allow it to be divided. In embodiments, the optical force and/or pressure may be applied in the opposite direction of the movement of the material through the channel, e.g., against the flow, or in the same direction as the movement of the material in the channel, e.g., with the flow. may be applied. Cell or particle sorting can be performed on a single device or on separate chips. For example, in FIG. 1B, the fifth channel before outlet tube 145 includes one or more branches to allow for single or multiple sorting areas.

In another aspect of the invention, a manifold is connected to a vial such that tubing passes through the manifold and connects to an opposing vial or other container that contacts the substance (eg, fluid) within the vial. The manifold allows the vial to be connected to the microfluidic chip but stored below the chip, and/or the manifold allows the contents of the vial to be injected from the bottom of the chip; Vials or tubing from vials connect to or communicate with the microfluidic chip, mitigating some of the problems experienced by prior art techniques, such as sedimentation of cells or particles.

In another aspect, the invention is directed to a microfluidic chip holder that includes a light source guiding structure that includes an integrated prism cavity, and when the prism is mated, the light is directed at an angle relative to the chip. It emits light. This is the preferred method for illuminating constrained geometries. In embodiments, the light source includes, but is not limited to, an optical fiber or a collimated or focused light source. This light source is specifically directed or oriented precisely towards the analysis channel.

In another aspect of the invention, the device includes a second imaging device oriented orthogonally to the first camera and channel view. The reasons for the second camera vary. In one embodiment, the reason for the second imaging device is to assist in visual alignment of the laser or optical force within the analysis channel. In another aspect, the methods described herein can be used to record data from a first camera. At the second camera, data can be combined with data from the first camera, resulting in additional data that can be used to more accurately extrapolate cell location, size, shape, volume, etc. . This additional information about the same cell (or particle) increases the accuracy and range of measurements and analyses. In a further aspect, a second camera is combined with the first camera to capture the cells or particles imaged by the orthogonal camera and the camera in the flow or toward the side of the chip. Allows 3D reconstruction of groups of particles. using an algorithm described herein or elsewhere, including reversing or slowing the flow and taking one or more images of a particular cell or particle, or group of cells or particles. , the present invention allows multiple images to be analyzed and processed. Thereby, it is possible to determine characteristics/attributes/quantitative measurements such as cell volume, cell shape, nuclear position, nuclear volume, organelle or inclusion body position. In another aspect of the invention, the camera is oriented to image an in-axis image in the direction of flow.

In one aspect, the present invention does not require serpentine or zigzag channels to keep particles properly suspended, as is preferred according to the prior art. Due to the vertical nature of the fluid and particles or cells injected into the chip, the zigzag channels are vertically integrated with direct flow through the channel into the first horizontal channel (herein referred to as the second channel). Avoided by pumped particles.

The accompanying drawings depict certain aspects of some embodiments of the invention and should not be used to limit or define the invention. The drawings, together with the written description, serve to explain certain principles of the invention.
FIG. 1A shows an overall view of the device configuration, including the chip, manifold, and vial. FIG. 1B is a diagram showing a specific depiction of the orientation and location of the chip channels. 2A and B contain diagrams showing a microfluidic chip holder according to the invention. 2A and B contain diagrams showing a microfluidic chip holder according to the invention. 3A and 3B are views showing angles and aspects of a tip holder according to the invention. 3A and 3B are views showing angles and aspects of a tip holder according to the invention. FIGS. 4A and 4B are diagrams illustrating an example of a cell path and imaging configuration for a chip. FIGS. 4A and 4B are diagrams illustrating an example of a cell path and imaging configuration for a chip. FIG. 4C is a diagram illustrating alternative cell paths. FIG. 5 is a diagram illustrating on-chip multiplane imaging and how it can be used to render 3D images and information. 6A and 6B are diagrams illustrating multi-planar imaging on a chip in fluid flow and how it can be used to depict 3D images. 6A and 6B are diagrams illustrating multi-planar imaging on a chip in fluid flow and how it can be used to depict 3D images. FIG. 7 shows how cells can be captured and/or equilibrated within the analysis portion of the chip and imaged from multiple angles. FIG. 8 shows how the camera and illumination source are positioned along the laser and flow direction so that the particles move away from the camera. FIG. 9 shows how the camera and illumination source can be positioned along the laser and flow direction so that the particles move towards the camera.

The invention has been described with reference to specific embodiments having various features. It will be apparent to those skilled in the art that various modifications and variations can be made in the practice of the invention without departing from the scope or spirit of the invention. Those skilled in the art will recognize that these features can be used alone or in any combination based on the requirements and specifications of a given application or design. Embodiments including various features may also consist of, or consist essentially of, these various features. Other embodiments of the invention will be apparent to those skilled in the art from consideration of this specification and practice of the invention. The description of the invention provided is merely exemplary or explanatory in nature and, therefore, variations that do not depart from the essence of the invention are intended to be within the scope of the invention.

Before describing at least one embodiment of the invention in detail, it is important to note that the invention is not limited in its application to the details of construction and arrangement of components that are set forth in the following description or shown in the drawings. I want you to understand. The invention is capable of other embodiments or of being practiced or carried out in various ways. It is also understood that the language and terminology used herein are for purposes of description and are not to be considered limiting.

Referring now to the drawings, FIG. 1A shows an overall view of the devices taught herein. Sample vials 130 are disposed below the microfluidic chip 100 (also commonly referred to herein as a substrate) and are not limited as to number or size of vials. The vial may contain one or more substances including, but not limited to, fluids, liquids, gases, plasma, serum, blood, cells, platelets, particles, etc., or combinations thereof. , configured to hold any type of sample that may be moved through the device. In the context of this specification, the term "fluid" or "sample" can be used generally to refer to one or more such substances. The vial is connected directly or indirectly using an air tube 110 in operative communication with the underside of the chip. They can also be connected by a manifold 120, as further shown in FIG. 1A. FIG. 1B shows a preferred embodiment of a microfluidic chip 100 in which substances, fluids, particles, and/or cells are injected into one external surface (e.g., the XZ plane in FIG. 1B), such as the edge of the microfluidic chip. . The implantation is shown in FIG. 1B along one of the planes shared by the minimum distance, such as the XZ plane or the XY plane. In this particular embodiment, the length of side X160 is shorter than the length of side Y170 and the length of side Z180. Such a configuration allows the sample to have minimal deviations when entering the channels on the chip (e.g. no right turn is required, as is the common case in the current state of the art).

In FIG. 1A, one or more vials 130 are connected to the microfluidic chip 100 so that substances, fluids, particles, and/or cells can be injected or pumped vertically and upwardly into the microfluidic chip. A manifold 120 is shown. The manifold allows for the injection of substances from the bottom of the chip, while the electronics, flow sensors, and tubing (both liquid and air) are arranged to minimize cell sedimentation, which optimizes throughput. The air tube provides pressure or vacuum, which, when sealed, provides a closed system within the manifold device. In one embodiment, there is no seal between the vial and the manifold, and there is a distance indicating that the pressure in the internal volume is atmospheric. This allows the substance to be pumped from or into a container that is open to the atmosphere (a vacuum is required for pumping from an open container). Position the electronics, flow sensors, and tubing (both liquid and air) such that the manifold optimizes throughput and minimizes cell sedimentation.

By injecting the contents from the bottom of the chip, the present invention minimizes horizontal movement of inlet tube 140 and outlet tube 145, which causes problems such as sedimentation of particles or cells within channel 150. The outlet tube is offset from the inlet tube in the Z and X dimensions (FIG. 1B) in this example. In embodiments, the outlet tube is offset rather than offset, straight, in-line, or at an angle with respect to the inlet tube. Such a configuration avoids the sedimentation that occurs in prior art techniques, such as when injected or pumped into the chip from the side in a horizontal direction, where the fluid combined with cells and/or particles must push the direction and fluid dynamics upwards. The current configuration eliminates the need for serpentine or zigzag vertical channels. Injection from the manifold and the bottom of the chip also allows additional elements, components, machinery, or hardware to be placed below the chip (see Figure 1A).

Manifold 120 functions by regulating air pressure above the contents within the vial and providing the correct geometry for the flow sensor and electronics. Tubing, such as fluid or air tubing, passes through the manifold and connects to the vial on the opposite side. Pressurized air passes through one side of the manifold, creating a closed pressurized system within the manifold. By adjusting the pressure in the confined area, the system allows changes to parameters such as flow rate and fluid dynamics. Pressurized areas are present in both vial 130 and manifold 120. In another embodiment, no air connection is required to the vial since the vial is open to the ambient atmosphere. This allows sampling of a greater variety of containers and sources. In such embodiments, vacuum is applied to one or more other vials such that a pressure differential is created to drive fluid flow from the open container.

In one embodiment, pressure-based sample injection is used. The vial is filled with sample in fluid and sealed with either a lid or tubing connected to the chip. The vial can be opened to air or sealed with a septum or other air-tight device before attaching the lid. In one aspect, the lid can include two connections, one for a fluid such as a gas and one for a liquid. Optionally, embodiments of the method further include providing a sample inlet line chip in communication with a sample inlet line in communication with the first channel.

In another embodiment, vacuum-based sample injection is used. The vial is filled with sample in fluid and sealed with either a lid or tubing connected to the chip. The vial can be opened to air or sealed with a septum before attaching the lid. In one aspect, the lid can include two connections, one for a fluid such as a gas and one for a liquid. If desired, fluid can be aspirated from a vial open to the atmosphere by applying vacuum pressure to one or more other vials. Optionally, embodiments of the method further include providing a sample inlet line chip in communication with a sample inlet line in communication with the first channel.

Tip holders 200, 300 are shown in FIGS. 2A-B and 3A-B. The chip holder comprises a structure for guiding a light source 210, such as a fiber optic light source, a light emitting diode, or a laser, and an integrated prism cavity 220, 320 in which a prism can be mounted. The light source is directed or aligned to the desired location by an integrated structure or channel 240 within the chip holder. The built-in space for a fiber optic light source allows illumination, such as the cone of illumination 250, even in a constrained geometric environment, such as on a microfluidic chip 230, 330 or a channel within a microfluidic chip. In preferred embodiments, this light source is specifically directed or directed or focused specifically at the analysis channel 260. In a preferred embodiment, the chip holder includes a built-in space for the prisms 220, 320 and a fiber optic light source 210 that allows illumination with a constrained geometry. The tip further includes holes or openings in the bottom of the holder 350 to accurately align the fluid tubes as described herein. In one embodiment, adjustable screws are integrated into threaded holes 360 on one or more surfaces for proper alignment.

FIG. 4A is a preferred embodiment of the microfluidic chip 400 described herein. As shown, fluid first travels vertically upward through the first channel 410 and then communicates with the second horizontal channel 420. Another vertical channel 430 takes fluid closer to the top of the chip, the point where the fourth channel 440 is horizontal, and contains the analysis channel, as shown in FIG. The channels are in operative communication with each other to allow the sample to move from one channel to another through the system. In embodiments, the sample can flow from the first channel to the second channel, to the third channel, to the fourth channel, or vice versa, or a combination thereof. A pump and/or vacuum device may be provided to provide positive and/or negative pressure at either or both the opening and outlet of the channel to enable movement of substances through the channel. The analysis channel is proximate one or more outer surfaces of the substrate, such as a face, edge, or side of the chip. For example, with this configuration, the analysis channels are in close proximity to the top and sides of the chip, thereby improving imaging and analysis through the material of the microfluidic chip. In preferred embodiments, the analysis channels are about 1 mm to about 2 mm from the top and sides of the chip. However, the distance of the analysis channel from the top of the chip can be from 0.1 mm to 100 mm, such as from 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm. Stated another way, the analysis channels can be located within the top 50%, 33%, 25%, 10%, or 5% of the substrate. The length of the horizontal analysis channel according to the present invention can be from about 250 microns to about 10 mm. However, the length of the analysis channel may be 100 microns to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm. In other words, the length of the analysis channel is no more than about 75% of the height, width, or length of the substrate/chip, such as no more than 50%, no more than 33% of the height, width, or length of the substrate/chip. , 25% or less, 10% or less, or 5% or less.

Two imaging devices 450, such as machine vision cameras, are shown in FIG. 4B. In one embodiment, a camera may be placed above the chip and oriented orthogonally to the analysis channel. In another embodiment, the camera is placed on the side of the chip, either perpendicular to the direction of flow or obliquely to the direction of flow (e.g., above the channel, below the channel, or against the side of the channel). may be oriented (at an angle). In embodiments, the camera is configured to image at any angle to the one or more material streams, such as perpendicular or 90 degrees to the one or more material streams, or from 0 to 90 degrees, or from 10 to It can be arranged to run at 80 degrees, or 30-60 degrees, etc. In another embodiment, two or more cameras can be used to image cells or particles within the analysis channel. For example, the camera may be oriented above the chip and orthogonally to the analysis channel. A second camera is placed on the side of the chip, either perpendicular to the direction of flow or oblique to the direction of flow (e.g., above the channel, below the channel, or at an angle to the side of the channel). ) may be oriented. As shown in FIG. 4B, one or more light sources 460 can be used to illuminate the analysis channel 440, such light sources being placed below the chip and illuminating the sides of the chip, in the flow direction or flow direction. It can be illuminated in the opposite direction, illuminated onto the chip, and illuminated diagonally into the analysis channel.

Alternatively, a dichroic mirror 840 or other suitable optical element can be used to selectively redirect light in certain wavelength ranges and pass light in other wavelength ranges, as shown in FIG. . This allows camera 810 to be placed in line with the analysis channel. Several embodiments occur, including placing the optical power laser 830 and camera 810 at the same end or opposite ends of the analysis region, as shown in FIGS. 8 and 9. Also, an illumination source 860 for the camera may be required and can be oriented in several ways, such as those shown in FIGS. 8 and 9. The light source can be a broad spectrum light source, such as one or more LEDs, or a narrow light source, such as a laser. The camera can be used in combination with other viewpoints, as a single camera, or as part of a multi-camera system, as described herein.

Also, as depicted in FIG. 4A, a light source 480, such as a laser, can be used to influence cell flow. The laser may be placed along the flow of cells or can be placed opposite the flow of cells. Also, the laser can be placed and/or oriented perpendicular or oblique to the cell flow.

One embodiment of the invention includes a device for particle analysis (see, eg, FIGS. 4-9). Embodiments of the invention include at least one camera 450 to capture images of particles or cells within the microfluidic channel (eg, 440). In one embodiment, a laser or other optical power 480 is included, such as a collimated light source operable to generate at least one collimated light source beam. The at least one collimated light source beam includes at least one beam cross section. Embodiments of the invention provide a method in which a first plane traverses the first channel 410 substantially along its length, whereby a fluid sample is injected into the substrate/chip from the bottom of the chip, under positive or negative pressure. It includes a substrate having a first channel 410 extending vertically within the substrate so as to be forced upwardly by the pressure. Embodiments of the invention are orthogonal to the first channel, such that the second plane traverses the second channel 420 substantially along its length, and the second plane is disposed orthogonal to the first plane. The second channel 420 is horizontally disposed within the substrate so that the second channel 420 is horizontally disposed within the substrate. This second channel is horizontal to the chip. The second channel communicates directly or indirectly with the first channel. The second channel communicates directly or indirectly with a short upward vertical third channel 430 that brings the channel network closer to the top of the chip. The third channel communicates directly or indirectly with a fourth horizontal channel 440 located near the top and/or corner of the chip. In a preferred embodiment, the fourth channel is the channel closest to the top of the chip. In one embodiment, the fourth channel is an analysis channel. In one aspect, camera 450 is oriented perpendicular to the flow direction within the fourth channel. Embodiments of the invention include a focused particle stream nozzle operably connected to the first channel. In another aspect of the invention, the second channel undergoes resizing and passes through the nozzle before communicating with the third channel.

FIG. 4C shows another embodiment of a sample path for a microfluidic chip. As shown here, fluid travels vertically upwardly within the first chip through a first channel 410, which then connects directly with a second horizontal channel 420, in this example at the top of the chip. or communicate indirectly, in this embodiment including an analysis channel. According to this configuration, the analysis channel is close to the top of the chip, thereby improving imaging and analysis through the material of the microfluidic chip. In preferred embodiments, the analysis channels are about 1 mm to about 2 mm from the top and sides of the chip. However, the distance of the analysis channel from the top of the chip can be from 0.1 mm to 100 mm, such as from 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm. Stated another way, the analysis channels can be located within the top 50%, 33%, 25%, 10%, or 5% of the substrate. The length of the horizontal analysis channel according to the present invention can be from about 250 microns to about 10 mm. However, the length of the analysis channel may be 100 microns to 100 mm, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm, 0.3 mm to 0.4 mm. In other words, the length of the analysis channel is no more than about 75% of the height, width, or length of the substrate/chip, such as no more than 50%, no more than 33% of the height, width, or length of the substrate/chip. , 25% or less, 10% or less, or 5% or less. In embodiments, the substrate can include one or more analysis channels, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 analysis channels.

Also, as depicted in FIG. 4C, a light source 480, such as a laser, can be used to influence the flow of matter, such as the flow of cells. The laser may be placed along the flow of cells or may be placed opposite the flow of cells. The laser may also be positioned and/or oriented perpendicular or oblique to the cell flow.

In Figures 1 and 4, a fluid stream containing cells or particles is directed vertically through a first channel. One or more substances (fluids, cells, and/or particles) enter through the bottom of the chip at the opening of the first channel and enter the substrate in a vertical direction. The first vertical channel is 100 microns to 100 mm in length, such as 0.1 mm to 0.2 mm, 0.2 mm to 0.3 mm. The first channel is followed by a second orthogonal/horizontal channel, which in a preferred embodiment is shorter than the first channel. The second channel may have a length of 250 microns to 100 mm, such as 0.25 mm to 0.5 mm, 0.5 mm to 0.75 mm, 0.75 mm to 1.0 mm. The third channel extends perpendicularly and parallel to the first channel. The third channel may have a length of 50 microns to 100 mm, such as 0.05 mm to 0.1 mm, 0.1 mm to 0.15 mm, 0.15 mm to 0.2 mm. The channels are placed in operative communication, directly or indirectly, to allow one or more substances to move through the plurality of channels. Typical directions of fluid flow are given by the flow arrows in FIGS. 4A and 4C, but may be reversed.

In preferred embodiments, the fourth channel comprises an analysis channel that is a channel with a length of 250 microns to 100 mm, such as 0.25 mm to 0.5 mm, 0.5 mm to 0.75 mm, 0.75 mm to 1.0 mm. In this embodiment, the fourth channel is the channel closest to the top of the chip. The distance of the fourth channel from the top of the chip, measured vertically, may be between 100 microns and 2 mm, but can be as large as 100 mm, such as between 100 microns and 200 microns, between 200 microns and 300 microns, and between 300 microns and 400 microns. It may be In other words, the fourth channel can be located within the top 50%, 33%, 25%, 10%, or 5% of the chip. The imaging device, such as a camera, is orthogonal to the fourth channel and approximately 100 microns to 2 mm from the fourth channel, such as 100 microns to 200 microns, 200 microns to 300 microns, 300 microns to 400 microns, from the fourth channel. The size can be about 100 mm.

In one embodiment, a laser or other light source is present with the focusing lens element. FIG. 4A depicts the present invention with laser 480 activated, emitting a laser beam, and directing the beam through a focusing lens element into fourth flow channel 440. The particles are aligned within the laser beam due to gradient forces that pull the particles toward the region of highest laser intensity. The laser scattering force causes particles to propagate in the direction of laser beam propagation (eg, from left to right in FIG. 4A).

In another embodiment, a second camera or image capture device (see 450) is provided in addition to the camera or image capture device oriented orthogonally to the fourth channel, here on the side of the analysis (or fourth) channel. orthogonally or at any angle to the fourth channel, for example as shown in FIG. 4B. Obtaining one image of each cell in orthogonal view allows multiple cell properties, e.g. size and shape, to be calculated in two dimensions, increasing the amount of information that can be captured for each cell. . This also allows calculation of volumetric properties of cells including total volume, shape, and gives insight into cells that are not symmetrical with respect to an axis parallel to the direction of flow (Z-axis as depicted in Figure 5). Probably. Using two or more cameras, a basic 3D model of the cell 550 can be constructed by combining orthogonal images using existing 3D reconstruction algorithms, such as diffraction theory methods or illumination rotation methods. . 3D models and analysis of 3D models allow more accurate analysis of cells, such as cell size, shape, orientation, and other quantitative and qualitative measurements regarding particles or cells in the fourth channel of the microfluidic chip Make it.

In FIG. 5, a portion of analysis channel 540 is shown from two different planes, such as the plane that can be imaged from an imaging device (see, eg, FIG. 4). In a first plane 520, a portion of a cell or particle 510 is imaged in a particular orientation. In the second plane 530, another part of the same cell or particle is imaged from a different perspective with another camera. This allows calculation of multiple cell properties and produces a matrix of data per cell per camera and a 3D representation of the cell or particle 550.

Image different slices of a cell as the cell is moved, e.g. can be converted into This is shown in Figure 6A. In FIG. 6A, a portion of a cell or particle is imaged at multiple points in time and/or space, from different directions and from different viewpoints. In this example, as the cells move and rotate through the focal plane, they appear as different sizes in successive images (such as four example images per plane from two different cameras, as shown in Figure 6A). Using 3D reconstruction algorithms allows for more complex 3D rendering 650 of particles or cells. From such renderings, certain attributes of cells or particles, such as cell size, volume, nuclear position and size, and other organelles, and measurements or contours of cell topography, can be extrapolated. Additionally, cell rotation is a function of optical force-based torque due to changes in biophysical or biochemical properties, including but not limited to refractive index, birefringence, or cell shape or morphology. can be measured.

FIG. 6B, for example, depicts chip multiplane imaging in which multiple images of the same cell are imaged over time as the cell moves through the focal plan. In such cases, the view of the cell is referred to in the art as a "slice" or "image slice," where the image slice is substantially the thickness of the optical plane being imaged. The thickness of the image plane or slice is determined, among other things, by the optical magnification of the imaging system. At higher magnifications, the working distance of the objective lens decreases, resulting in the need to have the lens closer to the cell or particle being imaged. In one embodiment, a laser or other optical force 670 can be used to affect the flow of cells within the analysis channel. In a preferred embodiment, the cells or particles are imaged either by moving the laser and/or camera, or by adjusting the flow(s), or by adjusting the position using hydrodynamic focusing. can be intentionally guided into or out of the focal plane of the channel for the purpose of structuring. For example, the laser source can be moved a distance 660 using, for example, a piezoelectric actuator or a linear electro-optic mechanical stage. This can be done on a cell-by-cell or population-by-population basis. Hydrodynamic focusing of cells can be altered to affect the initial position and trajectory of the cells. For example, the particles may be aligned or oriented in the focal plane within the laser beam due to gradient forces that pull the particles toward the region of highest laser intensity. The laser scattering force causes particles to propagate in the direction of laser beam propagation. See Figure 6B. Moving the laser, in this case in the X-axis, allows imaging of features in different parts of the cell, represented by disk 680. This may represent, for example, a nucleus, organelles, inclusion bodies, or other features of a cell or particle. The laser draws cells to their center as a result of gradient forces. Additionally, it is contemplated that more than one camera may improve detail and accuracy.

The embodiment of the invention shown in FIG. 7 is a static mode in which particles or cells 710 are stopped at a particular differential holding position by balancing optical forces 730 and fluid forces 735. Optical force may be applied by, for example, a laser or a collimated light source . Images can be taken with multiple aircraft, such as in FIGS. 5 and 6A-B. A flow sensor is used to measure the flow rate at which each particle stops in the flow for a given laser power. Since the optical and fluid forces are balanced, the fluid drag (ie, from flow velocity and channel dimensions) is equal to the optical force. In this way, the characteristics of each cell can be measured sequentially. Although not a high-throughput measurement system, this embodiment of the invention allows close observation and imaging of captured cells, as well as dynamic changes in optical power resulting from biochemical or biological changes in the cells. . Reagent streams containing chemicals, biochemicals, cells, or other standard biological materials can be introduced into the flow channels to interact with the captured cells. These dynamic processes can be quantitatively monitored by measuring changes in optical force during experiments on single cells or multiple cells.

In one embodiment, the camera or other imaging device is oriented and/or focused in the direction of and/or opposite the flow of the analysis channel so that it is in series and parallel to the flow of the analysis channel. For example, see FIGS. 8 and 9). FIG. 8 shows a camera 810 along an analysis channel 820 and a laser or collimated light source 830. A dichroic mirror or similar device 840 reflects laser light 835 away from the camera to prevent damage, but passes light 865 produced by illumination source 860 to enable imaging. The camera is oriented parallel to the fluid flow 870 such that the cells or particles 880 are directed away from the camera in one embodiment. The illumination source is oriented perpendicular to the channel and the laser. A second dichroic 845 that passes the laser light and reflects the illumination light is used to pass both the illumination light and the laser light through the channel. An alternative embodiment of this configuration switches the position of the laser and illumination source so that the laser is perpendicular to the channel and the illumination source is parallel to the channel. The second dichroic still directs both laser light and visible light through the channel.

An alternative implementation is shown in FIG. In this case, the camera or imaging device 910 is oriented such that the cells or particles 980 move towards the camera in the fluid stream 970. Thus, the camera and laser 930 are on the same side of the channel, while the illumination source 960 is on the opposite end of the channel. Two dichroics 940 and 945 are used to direct laser light 935 and illumination light 965 into the channel, direct the illumination light into the camera, and deflect the laser light away from the illumination source. An alternative embodiment of this configuration switches the position of the laser and camera so that the laser is perpendicular to the channel and the illumination source is parallel to the channel. A second dichroic 945 then directs the laser light through the channel and shines the light onto the camera.

Optionally, embodiments of the invention provide a standard TEM00 mode beam, a standard TEMOO mode beam, a standard TEM10 mode beam, a standard Hermitian-Gaussian beam mode, a standard Laguerre-Gaussian beam mode between the optical power source and the fourth channel. - further comprising at least one optical element operable to generate a beam mode, a Bessel beam, or a standard multimode beam; Optionally, the at least one optical element is a standard cylindrical lens, a standard axicon, a standard concave mirror, a standard toroidal mirror, a standard spatial light modulator, a standard acousto-optic modulator, a standard piezoelectric mirror array, a diffractive optical element, a standard quadrant. Includes a single wave plate and/or a standard half wave plate. Optionally, the source of optical power may include a standard circularly polarized beam, a standard linearly polarized beam, or a standard elliptically polarized beam.

Optionally, the device comprises a microfluidic channel, a laser light source focused by an optical system, and an electric field source operably connected to the microfluidic channel via an electrode to move particles in a liquid into a microfluidic channel. Flow through the channels and manipulate laser light and electric fields to jointly act on the particles within the microfluidic channels, thereby modifying their size, shape, refractive index, charge, charge distribution, charge mobility, dielectric constant, and Devices are implemented that separate particles based on/or deformability. In yet another embodiment, a device includes a microfluidic channel configured to provide a dielectrophoretic (DEP) field inside the channel via (1) an electrode system or (2) an insulator DEP system; a laser light source focused into the microfluidic channel by a system, flowing a plurality of particles in the liquid into the microfluidic channel, operating the laser light and the field together on the particles in the microfluidic channel to or modify their velocities, the DEP fields are linear or nonlinear. Another possible embodiment of the device is a microfluidic channel that includes an inlet and multiple outlets and that maximizes the residence time of selected particles within the laser light while exerting optical forces on the particles, thus the laser light is operable to apply a force to the particles flowing through the microfluidic channel, thereby increasing the velocity of the flow within the microfluidic channel to separate the particles into multiple outlets. and a laser light source optically focused across the microfluidic channel at a matched critical angle.

Optionally, embodiments of the invention further include at least one particle interrogation unit in communication with one or more of the analysis channel(s), in particular a fourth channel. The particle interrogation unit includes standard illuminators, standard optics, and standard sensors. Optionally, the at least one particle interrogation unit includes a standard bright field imaging device, a standard light scattering detector, a standard single wavelength fluorescence detector, a standard spectrofluorescence detector, a standard CCD camera, a standard CMOS camera, a standard photo diodes, standard photomultiplier tubes, standard photodiode arrays, standard chemiluminescent detectors, standard bioluminescent detectors, and/or standard Raman spectroscopic detectors.

At least one particle interrogation unit in communication with the fourth channel comprises a laser power-based device or devices that facilitate identification, selection, and sorting of cellular diseases. In one aspect, the unit utilizes inherent differences in optical pressure resulting from changes in particle size, shape, refractive index, or morphology as a means of particle separation and characterization. In one embodiment, a near-infrared laser beam exerts a physical force on the cell, which is then measured. When the optical force via radiation pressure is balanced against the fluid drag on the particles, it can be used to distinguish between different particles or to vary the particle velocity based on the inherent differences in the population of particles. Bring about change. A balance of fluid and optical forces can also be used to change the relative position of particles relative to each other based on their intrinsic properties, thereby providing physical separation. Another embodiment of the interrogation unit includes a device for particle analysis and/or separation, such as at least one collimated light source operable to generate at least one collimated light source beam. The at least one collimated light source beam includes at least one beam cross section.

One embodiment of the invention includes combining several of the design elements described above into a single device. Embodiments also include methods of using such devices. An example of such an integrated device is shown in FIG. Although the illustrated embodiment of the invention is a five-layer structure, with all five layers bonded to each other resulting in a solid-state microfluidic chip, the chip may also be a single structure as opposed to bonded layers. good. Chips include fused silica, crown glass, borosilicate glass, soda lime glass, sapphire glass, cyclic olefin polymer (COP), poly(dimethyl)siloxane (PDMS), OSTE, polystyrene, poly(methyl)methacrylate, polycarbonate, etc. Can be constructed using a number of standard materials including plastics or polymers. However, it is not limited to these. This chip allows clear optical access for sample input, hydrodynamic focusing, optical interrogation, imaging, analysis, sample exit, and laser light into and out of the region. Chips in embodiments can also be 3D printed, molded, or otherwise shaped.

Optionally, the at least one particle type includes multiple particle types. Each particle type of the plurality of particle types includes respective intrinsic properties and respective induced properties. Optionally, the intrinsic properties include size, shape, refractive index, morphology, intrinsic fluorescence, and/or aspect ratio. Optionally, the induced properties include deformation, angular orientation, rotation, rotational speed, antibody-labeled fluorescence, aptamer-labeled fluorescence, DNA-labeled fluorescence, stain-labeled fluorescence, differential retention metry, and/or gradient force metry. . Embodiments of the method further include identifying and separating the plurality of particles according to their respective particle types based on at least one of the intrinsic properties and the induced properties. Optionally, embodiments of the method further include interrogating or manipulating the sample flow. Optionally, examining the sample flow determines at least one of such intrinsic properties and induced properties of the particle type, and measuring particle velocities of the plurality of particles. including. The measurement of at least one of the endogenous properties may include determination of antibody or protein productivity for virus quantification, process development and monitoring, sample release assays, exogenous agent testing, clinical diagnostics, biomarker discovery, process development and monitoring; Determining the potency, quality, or activation state of cells produced as CAR T and other oncological applications and cell-based therapies, including stem cells, chemical, bacterial, viral, antimicrobial, or antimicrobial agents against specific cell populations. It can be used for a range of applications including, but not limited to, determining the effectiveness of viral agents, as well as determining disease status or potential of research or clinical cell samples. Optionally, the optical power source includes at least one beam axis and the sample flow includes a sample flow axis. Determining at least one of the intrinsic properties and induced properties of the particle type and jointly measuring particle velocities of the plurality of particles include offsetting the beam axis from the sample flow axis. Optionally, the step of determining at least one of an intrinsic property and an induced property of a particle type and the step of jointly measuring particle velocities of a plurality of particles include at least one beam axis from a sample flow axis. calculating slopes and trajectories of the plurality of particles deviating toward the direction of the particle.

Those skilled in the art will recognize that the disclosed features may be used alone, in any combination, or omitted based on the requirements and specifications of a given application or design. It is understood that when an embodiment refers to "comprising" a particular feature, the embodiment can alternatively "consist of" or "consist essentially of" any one or more of the features. I want to be Other embodiments of the invention will be apparent to those skilled in the art from consideration of this specification and practice of the invention.

In particular, it is noted that where a range of values is provided herein, each value between the upper and lower limits of the range is also specifically disclosed. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range. The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Variations that do not depart from the essence of the invention are intended to be included within the scope of the invention, and such specifications and illustrations are intended to be considered to be exemplary or illustrative in nature. It is. Further, all of the references cited in this disclosure are each individually incorporated herein by reference in their entirety, thus providing an efficient way to supplement the possible disclosure of the present invention; It is intended to provide background detailing as well as the level of skill in the art.

Claims (91)

A device comprising a substrate comprising a plurality of channels configured to transport one or more substances, and a collimated light source capable of interacting with the one or more substances, the device comprising:
The plurality of channels are
a first channel oriented perpendicularly upward relative to gravity along the Y-axis in the substrate;
a second channel in operative communication with the first channel and disposed horizontally within the substrate along the X-axis;
a third channel in communication with the second channel and oriented vertically with respect to gravity upwardly in the substrate along the Y-axis;
a fourth channel communicating with the third channel and disposed horizontally within the substrate along the X axis;
The first channel, the second channel, the third channel, and the fourth channel pass through the substrate from the first channel to the second channel, the third channel, and the fourth channel. arranged to provide a path for the movement of more than one substance;
the second channel and the fourth channel are shorter in length than the first channel , and the collimated light source is oriented to interact with the one or more substances in the fourth channel. ,
the first channel is oriented upwardly along the Y axis;
receiving said one or more substances by injection via upward pressure or vacuum driven flow against gravity through a bottom horizontal planar surface having an opening to said first channel in a vertical direction; consists of,
device. the one or more substances are injected into the first channel vertically disposed within the substrate through a bottom horizontal planar surface having an opening to the first channel;
the bottom horizontal plane surface has a smaller or equal surface area in the vertical direction compared to the surface area on the vertical plane of the chip to maintain directionality and volumetric continuity with the first channel;
A device according to claim 1. the first channel is disposed on an outer surface of the substrate, and the one or more substances enter vertically into the substrate;
2. The device of claim 1, comprising an opening arranged in such a way as to provide a path for vertical movement within the first channel. The one or more substances are vertically injected into the first channel vertically disposed within the first channel through a bottom horizontal planar surface having an opening to the first channel, so that the first channel maintain directional and volumetric continuity with
A device according to claim 1. the one or more substances through a bottom horizontal planar surface having an opening to the first channel and into the first channel vertically disposed within the first channel, the direction and volume with the first channel; Injected vertically to maintain continuity,
the first channel and the opening are shaped, sized, and oriented in a manner that maintains directional and volumetric continuity;
A device according to claim 1. the collimated light source is oriented to propagate in the direction of, opposite to, orthogonal to, or oblique to the movement of one or more substances in the fourth channel; 2. The device of claim 1. 2. The device of claim 1, wherein the fourth channel allows imaging and analysis of particles or cells in multiple focal planes. 2. The device of claim 1, wherein the fourth channel allows imaging and analysis of particles or cells in multiple focal planes during movement of the one or more substances. 2. The device of claim 1, wherein the fourth channel allows imaging and analysis of particles or cells during movement of the one or more substances. 2. The device of claim 1, wherein the fourth channel allows imaging and analysis of particles or cells from multiple angles and/or orientations during movement of the one or more substances. The fourth channel allows imaging and analysis of particles or cells from multiple focal planes, angles, and/or orientations by one or more imaging devices during movement of the one or more substances. 2. The device of claim 1. 2. The device of claim 1, further comprising one or more electrical, optical, and/or fluidic forces to move cells or particles within the one or more channels. 2. The device of claim 1, further comprising one or more electrokinetic, electrophoretic, and/or dielectrophoretic (DEP) forces for moving cells or particles in one or more channels. further comprising one or more imaging devices;
at least one of the imaging devices can be moved to change the focal plane imaged in the fourth channel;
A device according to claim 1. Cells or particles within the fourth channel can be moved by changes in optical and/or fluidic forces;
the area of the cell imaged in the fourth channel changes as the particles move;
A device according to claim 1. the fourth channel is located a distance from two or more outer surfaces of the substrate;
allowing imaging and analysis of multiple image slices of a cell or particle as the focal plane moves relative to the cell or particle;
A device according to claim 1. the fourth channel is spaced apart from two or more outer surfaces of the substrate;
enabling imaging and analysis of multiple image slices of the moving cells or particles as they move through the focal plane;
A device according to claim 1. the fourth channel is spaced apart from two or more outer surfaces of the substrate;
2. The device of claim 1, which allows imaging and analysis of multiple image slices of floating or stationary cells or particles. 2. The device of claim 1, wherein the one or more substances can be moved by pressure, vacuum, peristalsis, electrokinetic force, electrophoretic force, magnetic force, optical force, or any combination thereof. 2. The device of claim 1, further comprising a dichroic mirror for directing light to or away from an imaging device that images and/or analyzes cells or particles within the fourth channel. 2. The device of claim 1, further comprising a dichroic mirror for directing a collimated or focused light source to interact with cells or particles within the fourth channel. 2. The device of claim 1, further comprising a dichroic mirror for directing a collimated or focused light source away from an imaging device , another light source, or another portion of the device. 2. The device of claim 1, wherein the plurality of channels includes a fifth channel disposed horizontally, vertically, or diagonally within the substrate. 2. The device of claim 1, wherein the plurality of channels includes a fifth channel that is divided into two or more channels or wells for sorting cells or particles. 2. The device of claim 1, wherein the fourth channel is located closer to a top of the substrate than any of the first channel, the second channel, or the third channel. 2. The device of claim 1, wherein the fourth channel is located between 100 microns and 100 mm from the top of the substrate. 2. The device of claim 1, wherein the first channel has a length ranging from 0.1 mm to 100.0 mm. 2. The device of claim 1, wherein the second channel has a length ranging from 0.1 mm to 100.0 mm. 2. The device of claim 1, wherein the third channel has a length ranging from 0.05 mm to 100.0 mm. 2. The device of claim 1, wherein the fourth channel ranges in length from 0.1 mm to 100.0 mm. 2. The device of claim 1, wherein the first channel is longer in length than the second channel, the third channel, or the fourth channel. 2. The device of claim 1, further comprising a cell or particle interrogation unit. 2. The device of claim 1, further comprising a cell or particle collection channel. Bright field imagers, light scattering detectors, single wavelength fluorescence detectors, spectroscopic fluorescence detectors, CCD cameras, CMOS cameras, photodiodes, photodiode arrays (PDAs), spectrometers, photomultiplier tubes or tube arrays, photodiodes array, chemiluminescence detector, bioluminescence detector, standard Raman spectroscopic detection system, surface-enhanced Raman spectroscopy (SERS), coherent anti-Stokes Raman spectroscopy (CARS), and/or coherent Stokes Raman spectroscopy (CSRS). 2. The device of claim 1, further comprising an imaging device selected from at least one of: a tube for injecting the one or more substances into an opening disposed on an outer surface of the substrate to provide a path for the one or more substances to enter the substrate and travel within the first channel; further comprising an end;
A device according to claim 1. A device comprising a substrate having a plurality of internal fluidic channels and a collimated light source capable of interacting with moving cells and/or particles, the device comprising:
The plurality of internal fluid channels is a first channel of the plurality of internal channels oriented perpendicularly upward relative to gravity along the Y-axis with respect to the substrate, and wherein the cells or particles are a first channel flowing upwardly through the first channel along the length of the first channel;
disposed on an outer surface of the substrate and operable with the first channel such that one or more substances can enter and move vertically and upwardly relative to gravity within the first channel; an opening communicating with the
a second channel of the plurality of internal channels disposed in the substrate oriented horizontally along the X-axis with respect to the substrate and in operative communication with the first channel, the second channel or particles flow horizontally through a second channel along the length of the second channel, the second channel being shorter in length than the first channel, and the collimated light source is in the second channel. oriented to interact with the one or more substances, the first channel is oriented upwardly relative to gravity along the Y-axis and vertically oriented by injection via pressure or vacuum-driven flow. a second channel configured to receive said moving cells and/or particles upwardly relative to gravity through an opening in the second channel;
comprising a substrate;
A device for moving cells and/or particles in a fluid. The one or more substances are in directional and volumetric continuity with the first channel vertically disposed within the substrate through a bottom horizontal planar surface having an opening to the first channel. 37. The device of claim 36 , wherein the device is injected vertically to maintain stability. the one or more substances are injected into the first channel vertically disposed within the substrate through a bottom horizontal planar surface having an opening to the first channel;
the bottom horizontal plane surface has a smaller surface area in the vertical direction compared to the surface area on the vertical plane of the chip to maintain directionality and volumetric continuity with the first channel;
37. The device of claim 36 . The first channel is disposed on an outer surface of the substrate and is arranged to provide a path for the one or more substances to vertically enter the substrate and move vertically within the first channel. 37. The device of claim 36 , comprising an opening. The one or more substances are introduced into the first channel through a bottom horizontal planar surface having an opening to the first channel in a vertical direction to maintain directional and volumetric continuity with the first channel. injected into said first channel arranged perpendicular to
37. The device of claim 36 . The one or more substances are introduced into the first channel through a bottom horizontal planar surface having an opening to the first channel in a vertical direction to maintain directional and volumetric continuity with the first channel. injected into the first channel disposed perpendicularly to
the first channel and the opening are shaped, sized, and oriented in a manner that maintains directional and volumetric continuity;
37. The device of claim 36 . The collimated or focused light source is configured to propagate in the same direction as, opposite to, perpendicular to, or oblique to the direction of movement of the one or more materials in the second channel. 37. The device of claim 36 , wherein the device is oriented to . 37. The device of claim 36 , wherein the second channel allows imaging and analysis of particles or cells in multiple focal planes. 37. The device of claim 36 , wherein the second channel allows imaging and analysis of particles or cells in multiple focal planes during movement of the one or more substances. 37. The device of claim 36 , wherein the second channel allows imaging and analysis of particles or cells during movement of the one or more substances. 37. The device of claim 36 , wherein the second channel allows imaging and analysis of particles or cells from multiple angles and/or orientations during movement of the one or more substances. The second channel allows imaging and analysis of particles or cells from multiple focal planes, angles, and/or orientations by one or more imaging devices during the movement of the one or more substances. 37. The device of claim 36 . 37. The device of claim 36 , further comprising one or more electrical, optical, and/or hydrodynamic forces to move cells or particles within the one or more channels. 37. The device of claim 36 , further comprising one or more electrokinetic, electrophoretic, and/or dielectrophoretic (DEP) forces for moving cells or particles within the one or more channels. further comprising one or more imaging devices;
at least one of the imaging devices can be moved to change the focal plane imaged in the second channel;
37. The device of claim 36 . cells or particles within the second channel can be moved by changes in optical and/or fluidic forces;
the area of the cell imaged within the second channel changes as the particles move;
37. The device of claim 36 . The second channel is at a distance from two or more outer surfaces of the substrate, allowing imaging and analysis of multiple image slices of cells or particles as the focal plane moves relative to the cells or particles. 37. The device of claim 36, wherein the device is placed one at a time. the second channel is spaced apart from two or more outer surfaces of the substrate;
enabling imaging and analysis of multiple image slices of the moving cells or particles as they move through the focal plane;
37. The device of claim 36 . the second channel is spaced apart from two or more outer surfaces of the substrate;
allows imaging and analysis of multiple image slices of floating or stationary cells or particles,
37. The device of claim 36 . 37. The device of claim 36 , wherein the one or more substances can be moved by pressure, vacuum, peristalsis, electrokinetic forces, electrophoretic forces, magnetic forces, optical forces, or any combination thereof. 37. The device of claim 36 , further comprising a dichroic mirror for directing light to or away from an imaging device that images and/or analyzes cells or particles within the second channel. 37. The device of claim 36 , further comprising a dichroic mirror for directing the collimated light source to interact with cells or particles within the second channel. 37. The device of claim 36 , further comprising a dichroic mirror for directing the collimated light source away from an imaging device, another light source, or another portion of the device. 37. The device of claim 36 , wherein the plurality of internal fluid channels includes a third channel disposed horizontally, vertically, or diagonally within the substrate. 37. The device of claim 36 , wherein the plurality of internal fluidic channels includes a third channel that is divided into two or more channels or wells for sorting cells or particles. 37. The device of claim 36 , wherein the second channel is located closer to the top of the substrate than the first channel. 37. The device of claim 36 , wherein the second channel is located between 100 microns and 100 mm from the top of the substrate. 37. The device of claim 36 , wherein the first channel ranges in length from 0.1 mm to 100.0 mm. 37. The device of claim 36 , wherein the second channel ranges in length from 0.1 mm to 100.0 mm. 37. The device of claim 36 , wherein the first channel is longer in length than the second channel. 37. The device of claim 36 , further comprising a cell or particle interrogation unit. 37. The device of claim 36 , further comprising a cell or particle collection channel. Bright field imagers, light scattering detectors, single wavelength fluorescence detectors, spectroscopic fluorescence detectors, CCD cameras, CMOS cameras, photodiodes, photodiode arrays (PDAs), spectrometers, photomultiplier tubes or tube arrays, photodiodes arrays, chemiluminescent detectors, bioluminescent detectors, standard Raman spectroscopic detection systems, surface-enhanced Raman spectroscopy (SERS), coherent anti-Stokes Raman spectroscopy (CARS), and/or coherent Stokes Raman spectroscopy (CSRS). 37. The device of claim 36 , further comprising an imaging device selected from at least one of: the one or more substances enter the substrate;
further comprising a tube end for injecting the one or more substances into an opening disposed on an outer surface of the substrate to provide a path for travel within the first channel;
37. The device of claim 36 . In order to sort cells or particles into one or more distinct regions or wells, said one or more substances can be controlled by pressure, vacuum, peristalsis, electromotive force, electrophoretic force, magnetic force, optical force, or any of the above. 37. The device of claim 36 , wherein the device is capable of movement by a combination of: The one or more substances are moved by pressure, vacuum, peristalsis, electrokinetic forces, electrophoretic forces, magnetic forces, optical forces, or any combination thereof to separate the cells or particles into one or more discrete regions or 37. The device of claim 36 , capable of being sorted into wells. A method for evaluating biological particles for use in cellular immunotherapy, the method comprising:
a) injecting one or more biological particles into a first channel of a substrate comprising a plurality of channels configured to transport one or more biological particles, said plurality of channels teeth,
i) a first channel oriented vertically upward relative to gravity along the Y-axis in the substrate;
ii) a second channel disposed horizontally within the substrate along the X-axis and in operative communication with the first channel;
iii) a third channel in communication with the second channel and oriented vertically upward relative to gravity along the Y-axis with respect to the substrate;
iv) a fourth channel disposed horizontally in the substrate along the X-axis and in communication with the third channel;
b) evaluating the biological particles via optical force-based measurements in the fourth channel using a collimated light source capable of interacting with the biological particles;
the horizontal channel is shorter in length than the first channel;
The first channel, the second channel, the third channel, and the fourth channel are connected to the substrate from the first channel to the second channel to the third channel to the fourth channel. arranged in such a manner as to provide a path for movement of said one or more biological particles through;
Injecting the one or more biological particles via pressure or vacuum-driven flow into a first channel oriented perpendicularly upward to gravity along the Y-axis within the substrate; through a bottom horizontal plane having an opening to the first channel;
The biological particles include T cells, engineered T cells including CAR-T cells,
The optical force-based measurements include morphology, motility, binding affinity, binding profile, effects on other biological particles, effects on target cells, biological, biochemical, chemical, physical, used to obtain information about the sensitivity to external forces such as and temperature effects,
Assessing the biological particle includes characterization of T cell receptors, regulatory T cells, allogeneic T cells, or bionic T cells.
A method, including steps. The evaluation includes quantitative measurement of the amount of change in the T cells,
provide predictive or prescriptive analysis;
73. The method of claim 72 . 1 compared to at least one length from one edge to another on the vertical planar surface of the substrate to maintain directional and volumetric continuity with the first channel. 73. The method of claim 72 , having at least one short length from one edge to another. the one or more biological particles are injected into the first channel vertically disposed within the substrate through a bottom horizontal planar surface having an opening to the first channel;
The bottom horizontal plane surface has a surface area in the vertical direction compared to the surface area on the vertical plane of the chip.
having a smaller or equal surface area to maintain directional and volumetric continuity with the first channel;
73. The method of claim 72 . the first channel is located on an outer surface of the substrate;
an opening arranged in such a manner as to provide a path for the one or more biological particles to vertically enter the substrate and travel vertically within the first channel;
73. The method of claim 72 . 73. The method of claim 72 , further comprising a collimated or focused light source oriented to interact with biological particles or cells within the fourth channel. The collimated or focused light source is arranged in the direction of movement of the one or more biological particles in the fourth channel, in the opposite direction of said movement, in a direction perpendicular to said movement, or oblique to said movement. 73. The method of claim 72 , wherein the method is oriented to propagate. The fourth channel is configured to detect biological particles during movement of the one or more biological particles or cells and from multiple focal planes, angles, and/or orientations by the one or more imaging devices. 73. The method of claim 72 , wherein the method allows imaging and analysis of cells. 73. The method of claim 72 , further comprising one or more electrical, optical, and/or hydrodynamic forces to move biological particles or cells within the one or more channels. The plurality of channels include a fifth channel,
the fifth channel is divided into two or more channels or wells for sorting cells or particles;
73. The method of claim 72 . Bright field imagers, light scattering detectors, single wavelength fluorescence detectors, spectroscopic fluorescence detectors, CCD cameras, CMOS cameras, photodiodes, photodiode arrays (PDAs), spectrometers, photomultiplier tubes or tube arrays, photodiodes arrays, chemiluminescent detectors, bioluminescent detectors, standard Raman spectroscopic detection systems, surface-enhanced Raman spectroscopy (SERS), coherent anti-Stokes Raman spectroscopy (CARS), and/or coherent Stokes Raman spectroscopy (CSRS). 73. The method of claim 72 , further comprising an imaging device selected from at least one of: 73. The method of claim 72 , wherein LFC (laser power cytology) is used to evaluate the biological particles or cells. 84. The method of claim 83 , wherein use of the LFC includes a combination of microfluidics and light-induced pressure to make measurements including optical force, pressure, size, and velocity on a cell-by-cell basis. The use of LFC is used to characterize biological particles,
the properties include morphology, motility, interaction of the biological particle with other physiological components, deformability (cytoskeletal changes), or the response of the biological particle to environmental changes;
84. The method of claim 83 . 74. The method of claim 73 , wherein evaluating the biological particle comprises measuring a complex of one or more cell therapy product cells interacting with the one or more target cells or particles. A method for evaluating biological particles for use in cellular immunotherapy, the method comprising:
a) injecting one or more biological particles into a first channel of a substrate comprising a plurality of channels configured to transport one or more biological particles;
The plurality of channels are:
i) a first channel oriented vertically upward along the Y axis in the substrate;
ii) a second channel disposed horizontally within the substrate along the X-axis and in operative communication with the first channel;
iii) a third channel oriented perpendicularly upward relative to gravity along the Y-axis in the substrate and in communication with the second channel;
(iv) a fourth channel disposed horizontally within the substrate along the X-axis and communicating with the third channel;
steps, including;
b) evaluating said biological particles for use in cellular immunotherapy through optical force-based measurements using a collimated light source capable of interacting with said biological particles in said fourth channel; A step,
the horizontal channel is shorter in length than the first channel;
the first channel, the second channel, the third channel, and the fourth channel pass through the substrate from the first channel to the second channel, the third channel, and the fourth channel; arranged to provide a pathway for movement of the one or more biological particles;
a first channel oriented perpendicularly upward relative to gravity along the Y-axis within the substrate through a bottom horizontal planar surface having an opening to the first channel via pressure or vacuum driven flow; injecting the one or more biological particles into the channel;
The biological particles include T cells, engineered T cells, include CAR T cells, and the biological particles are evaluated through LFC (laser power cytology) measurement;
The LFC measurements include morphology, motility, binding affinity, binding profile, effects on other biological particles, effects on target cells, biological, biochemical, physical, and temperature effects. used to obtain information about susceptibility to external forces,
Assessing the biological particle includes characterization of T cell receptors, regulatory T cells, allogeneic T cells, or bionic T cells.
A method, including steps. 88. The method of claim 87 , wherein the use of LFC includes a combination of microfluidics and light-induced pressure to make measurements including optical force, pressure, size, and velocity on a cell-by-cell basis. the use of LFC is used to characterize biological particles;
The properties include morphology, motility, interaction of the biological particle with other physiological components, deformability (cytoskeletal changes), or the response of the biological particle to environmental changes.
88. The method of claim 87 . 88. The method of claim 87 , wherein said evaluating a biological particle comprises measuring a complex of one or more cell therapy product cells interacting with one or more target cells or particles. Said evaluation of biological particles includes cell characterization for efficacy assays;
Claims including cell characterization for potency assays, CAR-T cell affinity testing, CAR-T cell binding testing, detection of T cell biomarkers, or label-free detection of T cell activation. The method according to item 87 .

Images